2GIG

Alteration of sequence specificity of the type II restriction endonuclease HINCII through an indirect readout mechanism


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.83 Å
  • R-Value Free: 0.220 
  • R-Value Work: 0.177 
  • R-Value Observed: 0.177 

wwPDB Validation   3D Report Full Report


This is version 1.6 of the entry. See complete history


Literature

Alteration of Sequence Specificity of the Type II Restriction Endonuclease HincII through an Indirect Readout Mechanism.

Joshi, H.K.Etzkorn, C.Chatwell, L.Bitinaite, J.Horton, N.C.

(2006) J Biol Chem 281: 23852-23869

  • DOI: https://doi.org/10.1074/jbc.M512339200
  • Primary Citation of Related Structures:  
    2GIE, 2GIG, 2GIH, 2GII, 2GIJ

  • PubMed Abstract: 

    The functional and structural consequences of a mutation of the DNA intercalating residue of HincII, Q138F, are presented. Modeling has suggested that the DNA intercalation by Gln-138 results in DNA distortions potentially used by HincII in indirect readout of its cognate DNA, GTYRAC (Y = C or T, R = A or G) (Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47). Kinetic data presented here indicate that the mutation of glutamine 138 to phenylalanine (Q138F) results in a change in sequence specificity at the center two base pairs of the cognate recognition site. We show that the preference of HincII for cutting, but not binding, the three cognate sites differing in the center two base pairs has been altered by the mutation Q138F. Five new crystal structures are presented including Q138F HincII bound to GTTAAC and GTCGAC both with and without Ca2+ as well as the structure of wild type HincII bound to GTTAAC. The Q138F HincII/DNA structures show conformational changes in the protein, bound DNA, and at the protein-DNA interface, consistent with the formation of adaptive complexes. Analysis of these structures and the effect of Ca2+ binding on the protein-DNA interface illuminates the origin of the altered specificity by the mutation Q138F in the HincII enzyme.


  • Organizational Affiliation

    Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA.


Macromolecules

Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
Type II restriction enzyme HincIIC [auth A],
D [auth B]
257Haemophilus influenzaeMutation(s): 1 
Gene Names: hincIIR
EC: 3.1.21.4
UniProt
Find proteins for P17743 (Haemophilus influenzae)
Explore P17743 
Go to UniProtKB:  P17743
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP17743
Sequence Annotations
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  • Reference Sequence

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Entity ID: 1
MoleculeChains LengthOrganismImage
5'-D(*GP*CP*CP*GP*GP*TP*CP*GP*AP*CP*CP*GP*GP*C)-3'A [auth E],
B [auth F]
14N/A
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.83 Å
  • R-Value Free: 0.220 
  • R-Value Work: 0.177 
  • R-Value Observed: 0.177 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 50.42α = 90
b = 90.99β = 104.1
c = 67.33γ = 90
Software Package:
Software NamePurpose
Blu-Icedata collection
SCALAdata scaling
CNSrefinement
CCP4data scaling
CNSphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2006-07-18
    Type: Initial release
  • Version 1.1: 2008-05-01
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2014-04-16
    Changes: Refinement description
  • Version 1.4: 2017-11-22
    Changes: Database references
  • Version 1.5: 2021-10-20
    Changes: Database references, Derived calculations
  • Version 1.6: 2023-08-30
    Changes: Data collection, Refinement description